Increasing the Complexity of Microtubule-based transport: Cargo adaptors and Hitchhiking on Vesicles.

PROJECT SUMMARY Proper positioning of intracellular cargos (organelles, vesicles, and macromolecules) is critical for cell growth, maturation, and survival. Eukaryotic cells use the molecular motors dynein and kinesin to transport cargos along microtubule tracks. Defects in microtubule-based transport and mutations in the motors themselves, are an underlying feature of many neurodevelopmental and neurodegenerative diseases. Since each type of cargo is tuned for specific cellular functions, their loading, movement and unloading along microtubules requires divergent transport mechanisms. How is cargo specificity on microtubules achieved? This proposal will address this with an in-depth analysis of the two major modes of microtubule-based transport. The canonical view, known as the cargo adaptor mode, is that each cargo interacts with specific adaptor proteins capable of recruiting molecular motors. The regulatory mechanisms by which cargos selectively load and unload from these cargo adaptors are not well understood. One goal of this proposal is to determine how a conserved phosphorylation site on vesicle-bound Rab GTPases affects interactions with dynein cargo adaptors and ultimately, how these interactions affect cellular function. Importantly, this Rab phosphorylation site is the predominant target of the Parkinson’s disease-linked kinase LRRK2. The other mode of transport is called organelle hitchhiking. In hitchhiking, Rab-vesicles can direct the movement of organelles on microtubules. To accomplish this, organelles attach to (or ‘hitchhike’ on) motor-driven Rab-vesicles at membrane contact sites. Organelle hitchhiking is a new, relatively unexplored paradigm of microtubule-based transport, and the underlying molecular mechanisms are not well understood. What are the molecular linkers, tethers, and regulators of organelle hitchhiking? Is this a prominent mode of microtubule-based transport? This proposal will use genetics, microscopy, and biochemistry in both fungal and mammalian model systems to address these questions. Taken together, studying the two main modes of microtubule-based transport is extremely important, given the critical roles Rabs, vesicles, and hitchhiking cargos play in development and age-related diseases.